Dose-Levels and First Signs of Efficacy in Contemporary Oncology Phase 1 Clinical Trials

PURPOSE: Phase 1 trials play a crucial role in oncology by translating laboratory science into efficient therapies. Molecular targeted agents (MTA) differ from traditional cytotoxics in terms of both efficacy and toxicity profiles. Recent reports suggest that higher doses are not essential to produce the optimal anti-tumor effect. This study aimed to assess if MTA could achieve clinical benefit at much lower dose than traditional cytotoxics in dose seeking phase 1 trials. PATIENTS AND METHODS: We reviewed 317 recent phase 1 oncology trials reported in the literature between January 1997 and January 2009. First sign of efficacy, maximum tolerated dose (MTD) and their associated dose level were recorded in each trial. RESULTS: Trials investigating conventional cytotoxics alone, MTA alone and combination of both represented respectively 63.0% (201/317), 23.3% (74/317) and 13.7% (42/317) of all trials. The MTD was reached in 65.9% (209/317) of all trials and was mostly observed at the fifth dose level. First sign of efficacy was less frequently observed at the first three dose-levels for MTA as compared to conventional cytotoxics or combinations regimens (48.3% versus 63.2% and 61.3%). Sign of efficacy was observed in the same proportion whatever the treatment type (73-82%). MTD was less frequently established in trials investigating MTA alone (51.3%) or combinations (42.8%) as compared to conventional cytotoxic agents (75.6%). CONCLUSION: First sign of efficacy was less frequently reported at the early dose-levels and MTD was less frequently reached in trials investigating molecular targeted therapy alone. Similar proportion of trials reported clinical benefit

Mitochondrial DNA common deletion is not associated with thyroid, breast and colorectal tumors in Turkish patients

Recently, efforts have been focused on mitochondrial DNA changes and their relation to human cancers. Among them, a 4977 bp deletion of mitochondrial DNA, named “common deletion”, has been investigated in several types of tumors, with inconsistent results. In this study, we investigated the presence of the common deletion in tissues from 25 breast, 25 colorectal and 50 thyroid tumors and in the adjacent healthy tissues from Turkish patients. Samples from healthy volunteers were also evaluated for comparison. Two PCR-based methods were used for the detection of the common deletion. First, two pairs of primers were used to amplify wild-type and deleted mtDNA. Then, a highly sensitive nested-PCR was performed, to determine low amounts of deleted genomes. By the first method, wild-type mtDNAs were observed in all samples, but a deletion was observed in only six thyroid samples, by using the nested-PCR method. In conclusion, the mitochondrial common deletion was very rare in our study group and did not appear to be not related with cancer

Mitochondrial DNA Copy Number Is Associated with Breast Cancer Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood is associated with increased risk of several cancers. However, data from prospective studies on mtDNA copy number and breast cancer risk are lacking. We evaluated the association between mtDNA copy number in peripheral blood and breast cancer risk in a nested case-control study of 183 breast cancer cases with pre-diagnostic blood samples and 529 individually matched controls among participants of the Singapore Chinese Health Study. The mtDNA copy number was measured using real time PCR. Conditional logistic regression analyses showed that there was an overall positive association between mtDNA copy number and breast cancer risk (Ptrend = 0.01). The elevated risk for higher mtDNA copy numbers was primarily seen for women with <3 years between blood draw and cancer diagnosis; ORs (95% CIs) for 2nd, 3rd, 4th, and 5th quintile of mtDNA copy number were 1.52 (0.61, 3.82), 2.52 (1.03, 6.12), 3.12 (1.31, 7.43), and 3.06 (1.25, 7.47), respectively, compared with the 1st quintile (Ptrend = 0.004). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥3 years before breast cancer diagnosis (Ptrend = 0.41). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in breast cancer risk. © 2013 Thyagarajan et al

A novel approach for rapid screening of mitochondrial D310 polymorphism

BACKGROUND: Mutations in the mitochondrial DNA (mtDNA) have been reported in a wide variety of human neoplasms. A polynucleotide tract extending from 303 to 315 nucleotide positions (D310) within the non-coding region of mtDNA has been identified as a mutational hotspot of primary tumors. This region consists of two polycytosine stretches interrupted by a thymidine nucleotide. The number of cytosines at the first and second stretches are 7 and 5 respectively, according to the GeneBank sequence. The first stretch exhibits a polymorphic length variation (6-C to 9-C) among individuals and has been investigated in many cancer types. Large-scale studies are needed to clarify the relationship between cytosine number and cancer development/progression. However, time and money consuming methods such as radioactivity-based gel electrophoresis and sequencing, are not appropriate for the determination of this polymorphism for large case-control studies. In this study, we conducted a rapid RFLP analysis using a restriction enzyme, BsaXI, for the single step simple determination of 7-C carriers at the first stretch in D310 region. METHODS: 25 colorectal cancer patients, 25 breast cancer patients and 41 healthy individuals were enrolled into the study. PCR amplification followed by restriction enzyme digestion of D310 region was performed for RFLP analysis. Digestion products were analysed by agarose gel electrophoresis. Sequencing was also applied to samples in order to confirm the RFLP data. RESULTS: Samples containing 7-C at first stretch of D310 region were successfully determined by the BsaXI RFLP method. Heteroplasmy and homoplasmy for 7-C content was also determined as evidenced by direct sequencing. Forty-one percent of the studied samples were found to be BsaXI positive. Furthermore, BsaXI status of colorectal cancer samples were significantly different from that of healthy individuals. CONCLUSION: In conclusion, BsaXI RFLP analysis is a simple and rapid approach for the single step determination of D310 polymorphism of mitochondrial DNA. This method allows the evaluation of a significant proportion of samples without the need for sequencing- and/or radioactivity-based techniques

Lack of association between mutations of gene-encoding mitochondrial D310 (displacement loop) mononucleotide repeat and oxidative stress in chronic dialysis patients in Taiwan

<p>Abstract</p> <p>Background</p> <p>Mitochondria (mt) are highly susceptible to reactive oxygen species (ROS). In this study, we investigated the association between a region within the displacement loop (D-loop) in mtDNA that is highly susceptible to ROS and oxidative stress markers in chronic dialysis patients. We enrolled 184 chronic dialysis patients and 213 age-matched healthy subjects for comparison. Blood levels of oxidative stress markers, such as thiobarbituric acid reactive substances (TBARS) and free thiol, and the mtDNA copy number were determined. A mononucleotide repeat sequence (CCCC...CCCTCCCCCC) between nucleotides 303 and 316-318 (D310) was identified in mtDNA.</p> <p>Results</p> <p>Depending on alterations in the D310 mononucleotide repeat, subjects were categorized into 4 subgroups: 7-C, 8-C, 9 or 10-C, and T-to-C transition. Oxidative stress was higher in chronic dialysis patients, evidenced by higher levels of TBARS and mtDNA copy number, and a lower level of free thiol. The distribution of 7-C, 8-C, and 9-10C in dialysis and control subjects was as follows: 7-C (38% <it>vs. </it>31.5%), 8-C (35.3% <it>vs. </it>43.2%), and 9-10C (24.5% <it>vs. </it>22.1%). Although there were significant differences in levels of TBARS, free thiol, and the mtDNA copy number in the D310 repeat subgroups (except T-to-C transition) between dialysis patients and control subjects, post hoc analyses within the same study cohort revealed no significant differences.</p> <p>Conclusion</p> <p>Although oxidative stress was elevated in chronic dialysis patients and resulted in a compensatory increase in the mtDNA copy number, homopolymeric C repeats in the mtDNA region (D310), susceptible to ROS, were not associated with oxidative stress markers in these patients.</p

Control region mutations and the 'common deletion' are frequent in the mitochondrial DNA of patients with esophageal squamous cell carcinoma

BACKGROUND: North central China has some of the highest rates of esophageal squamous cell carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA) accumulates more mutations than nuclear DNA and because of its high abundance has been proposed as a early detection device for subjects with cancer at various sites. We wished to examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area of China. METHODS: We used DNA samples isolated from tumors, adjacent normal esophageal tissue, and blood from 21 esophageal squamous cell carcinoma cases and DNA isolated from blood from 23 healthy persons. We completely sequenced the control region (D-Loop) from each of these samples and used a PCR assay to assess the presence of the 4977 bp common deletion. RESULTS: Direct DNA sequencing revealed that 7/21 (33%, 95% CI = 17–55%) tumor samples had mutations in the control region, with clustering evident in the hyper-variable segment 1 (HSV1) and the homopolymeric stretch surrounding position 309. The number of mutations per subject ranged from 1 to 16 and there were a number of instances of heteroplasmy. We detected the 4977 bp 'common deletion' in 92% of the tumor and adjacent normal esophageal tissue samples examined, whereas no evidence of the common deletion was found in corresponding peripheral blood samples. CONCLUSIONS: Control region mutations were insufficiently common to warrant attempts to develop mtDNA mutation screening as a clinical test for ESCC. The common deletion was highly prevalent in the esophageal tissue of cancer cases but absent from peripheral blood. The potential utility of the common deletion in an early detection system will be pursued in further studies

Suppression of adenine nucleotide translocase-2 by vector-based siRNA in human breast cancer cells induces apoptosis and inhibits tumor growth in vitro and in vivo

INTRODUCTION: Adenine nucleotide translocator (ANT) 2 is highly expressed in proliferative cells, and ANT2 induction in cancer cells is known to be directly associated with glycolytic metabolisms and carcinogenesis. In addition, ANT2 repression results in the growth arrest of human cells, implying that ANT2 is a candidate for cancer therapy based on molecular targeting. METHODS: We utilized an ANT2-specific RNA interference approach to inhibit ANT2 expression for evaluating its antitumor effect in vitro and in vivo. Specifically, to investigate the therapeutic potential of ANT2 repression, we used a DNA vector-based RNA interference approach by expressing shRNA to knockdown ANT2 in breast cancer cell lines overexpressing ANT2. RESULTS: ANT2 shRNA treatment in breast cancer cell line MDA-MB-231 repressed cell growth as well as proliferation. In addition, cell cycle arrest, ATP depletion and apoptotic cell death characterized by the potential disruption of mitochondrial membrane were observed from the ANT2 shRNA-treated breast cancer cells. Apoptotic breast cancer cells transfected with ANT2 shRNA also induced a cytotoxic bystander effect that generates necrotic cell death to the neighboring cells. The intracellular levels of TNFalpha and TNF-receptor I were increased in ANT2 shRNA transfected cells and the bystander effect was partly blocked by anti-TNFalpha antibody. Ultimately, ANT2 shRNA effectively inhibited tumor growth in vivo. CONCLUSION: These results suggest that vector-based ANT2 RNA interference could be an efficient molecular therapeutic method for breast cancer with high expression of ANT2.This work was supported in part by the grants from the Cancer Research Center, and the Korean Science & Engineering Foundation through the Tumor Immunity Medical Research Center at Seoul National University College of Medicine

Facile whole mitochondrial genome resequencing from nipple aspirate fluid using MitoChip v2.0

<p>Abstract</p> <p>Background</p> <p>Mutations in the mitochondrial genome (mtgenome) have been associated with many disorders, including breast cancer. Nipple aspirate fluid (NAF) from symptomatic women could potentially serve as a minimally invasive sample for breast cancer screening by detecting somatic mutations in this biofluid. This study is aimed at 1) demonstrating the feasibility of NAF recovery from symptomatic women, 2) examining the feasibility of sequencing the entire mitochondrial genome from NAF samples, 3) cross validation of the Human mitochondrial resequencing array 2.0 (MCv2), and 4) assessing the somatic mtDNA mutation rate in benign breast diseases as a potential tool for monitoring early somatic mutations associated with breast cancer.</p> <p>Methods</p> <p>NAF and blood were obtained from women with symptomatic benign breast conditions, and we successfully assessed the mutation load in the entire mitochondrial genome of 19 of these women. DNA extracts from NAF were sequenced using the mitochondrial resequencing array MCv2 and by capillary electrophoresis (CE) methods as a quality comparison. Sequencing was performed independently at two institutions and the results compared. The germline mtDNA sequence determined using DNA isolated from the patient's blood (control) was compared to the mutations present in cellular mtDNA recovered from patient's NAF.</p> <p>Results</p> <p>From the cohort of 28 women recruited for this study, NAF was successfully recovered from 23 participants (82%). Twenty two (96%) of the women produced fluids from both breasts. Twenty NAF samples and corresponding blood were chosen for this study. Except for one NAF sample, the whole mtgenome was successfully amplified using a single primer pair, or three pairs of overlapping primers. Comparison of MCv2 data from the two institutions demonstrates 99.200% concordance. Moreover, MCv2 data was 99.999% identical to CE sequencing, indicating that MCv2 is a reliable method to rapidly sequence the entire mtgenome. Four NAF samples contained somatic mutations.</p> <p>Conclusion</p> <p>We have demonstrated that NAF is a suitable material for mtDNA sequence analysis using the rapid and reliable MCv2. Somatic mtDNA mutations present in NAF of women with benign breast diseases could potentially be used as risk factors for progression to breast cancer, but this will require a much larger study with clinical follow up.</p